Streptococcus pneumoniae 37-kDa surface adhesin a protein

ABSTRACT

The invention provides a nucleic acid encoding the 37-kDa protein from  Streptococcus pneumoniae.  Also provided are isolated nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. The invention also provides purified polypeptides encoded by the nucleic acid encoding the 37-kDa protein from and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Also provided are antibodies which selectively binds the polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Also provided are vaccines comprising immunogenic polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. Further provided is a method of detecting the presence of  Streptococcus pneumoniae  in a sample comprising the steps of contacting a sample suspected of containing  Streptococcus pneumoniae  with nucleic acid primers capable of hybridizing to a nucleic acid comprising a portion of the nucleic acid encoding the 37-kDa protein, amplifying the nucleic acid and detecting the presence of an amplification product, the presence of the amplification product indicating the presence of  Streptococcus pneumoniae  in the sample. Further provided are methods of detecting the presence of  Streptococcus pneumoniae  in a sample using antibodies or antigens, methods of preventing and treating  Streptococcus pneumoniae  infection in a subject.

[0001] This application is a continuation-in-part of Ser. No.08/222,179, filed Apr. 4, 1994, which is a continuation-in-part of Ser.No. 07/791,377, filed Sep. 17, 1991, now U.S. Pat. No. 5,422,427, bothof which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the 37-kDa Streptococcus pneumoniaesurface adhesin A protein. Specifically, the invention relates to anisolated nucleic acid encoding the 37-kDa protein of Streptococcuspneumoniae, to unique fragments of the nucleic acid encoding the 37-kDaprotein of Streptococcus pneumoniae, and to the polypeptides encoded bythose nucleic acids. The invention further relates to antibodies tothose polypeptides, and to methods of detecting the presence ofStreptococcus pneumoniae, methods of preventing Streptococcus pneumoniaeinfection, and methods of treating a Streptococcus pneumoniae infection.

[0004] 2. Background Art

[0005] Pneumococcal disease continues to be a leading cause of sicknessand death in the United States and throughout the world. Both the lackof efficacy of the currently used polysaccharide vaccines in childrenunder 2 years of age and their variable serotype-specific efficacy amongvaccinated individuals, have prompted manufacturers to investigatealternative vaccine formulations that do not require the use of multiplecapsular polysaccharides. One current approach under consideration isthe use of immunogenic species-common proteins as vaccine candidates.These proteins could be used in combination with other immunogenicproteins or as protein carriers in a protein-polysaccharide oroligosaccharide conjugate vaccine. An effective vaccine that included acommon protein could eliminate the need for formulations based onmultiple capsular polysaccharides (as in the 23-valent polysaccharidevaccine) by offering a broader range of protection against a greaternumber of serotypes. Additionally, a protein-based vaccine would beT-cell dependent and provide a memory response, resulting in a moreefficacious vaccine.

[0006] Of the reported pneumococcal proteins, only pneumolysin and thepneumococcal surface protein A (PspA) have been extensively examined fortheir suitability as vaccine candidates. While both have been shown tobe partially protective in mice (Paton et al. 1983. “Effect ofimmunization with pneumolysin on survival time of mice challenged withStreptococcus pneumoniae.” Infect. Immun. 40:548-552 and McDaniel et al.1991. “PspA, a surface protein of Streptococcus Pneumoniae, is capableof eliciting protection against pneumococci of more than one capsulartype.” Infect. Immun. 59:222-228), there are disadvantages to their useas vaccine immunogens. Pneumolysin, although well conserved amongpneumococci, has been shown to have strong toxic effects in its nativestate (AlonsoDeVelasco et al. 1995. “Streptococcus pneumoniae: Virulencefactors, pathogenesis, and vaccines.” Microbiol. Rev. 59:591-603).Recombinant derivatives of reduced toxicity have been produced, andwhile they show promise in animal protection studies (Alexander et al.1994. “Immunization of nice with pneumolysin toxoid confers asignificant degree of protection against at least nine serotypes ofStreptococcus pneumoniae. Infect. Immun. 62:5683-5688) the problem ofmaintaining maximal immunogenicity and eliminating toxicity to humans isstill in question. PspA, on the other hand, is serologically andstructurally heterogeneous. (Crain et al. 1990. “Pneumococcal surfaceprotein A (PspA) is serologically highly variable and is expressed byall clinically important capsular serotypes of Streptococcuspneumoniae.” Infect. Immun. 58:3293-3299). Its use in vaccineformulations would require multiple PspA types, thus increasing thecomplexity of vaccine preparation.

[0007] An immunogenic species-common protein has been identified fromStreptococcus pneumoniae (Russell et al. 1990. “Monoclonal antibodyrecognizing a species-specific protein from Streptococcus pneumoniae.”J. Clin. Microbiol. 28:2191-2195 and U.S. Pat. No. 5,422,427 in whichthe 37-kDa protein is referred to as pneumococcal fimbrial protein A).The 37-kDa S. pneumoniae protein has been the focus of several studiesand has been designated pneumococcal surface adhesin protein A (PsaA).Immunoblot analysis studies using anti-PsaA monoclonal antibody showedthat PsaA is common to all 23 pneumococcal vaccine serotypes (Russell etal. 1990). Enzyme-linked-immunosorbent assay studies have indicated thatpatients with pneumococcal disease show an antibody increase inconvalescent-phase serum to PsaA compared with acute-phase serumantibody levels (Tharpe et al. 1995. “Purification and seroreactivity ofpneumococcal surface adhesin A (PsaA).” Clin. Diagn. Lab. Immunol.3:227-229 and Tharpe et al. 1994. “The utility of a recombinant proteinin an enzyme immunoassay for antibodies against Streptococcuspneumoniae.” abstr. V-2, p. 617. 1994. American Society forMicrobiology, Washington, D.C.). Additionally, a limited in vivoprotection study showed that antibodies to the 37-kDa protein protectmice from lethal challenge (Talkington et al. 1996. “Protection of miceagainst fatal pneumococcal challenge by immunization with pneumococcalsurface adhesin A (PsaA).” Microbial Pathogenesis 21:17-22).

[0008] The gene encoding PsaA from S. pneumoniae strain R36A (anunencapsulated strain) has been cloned in Escherichia coli andsequenced, but this serotype does not contain a 37 kDa protein encodingnucleic acid that is highly conserved among the various serotypes.(Sampson et al. 1994. “Cloning and nucleotide sequence analysis of psaA,the Streptococcus pneumoniae gene encoding a 37-kilodalton proteinhomologous to previously reported Streptococcus sp. adhesins.” Infect.Immun. 62:319-324). This particular nucleic acid and polypeptide,therefore, are of limited value for use as diagnostic reagents, ininfection prevention, in infection treatment, or in vaccine development.

[0009] Sequence conservation is a necessary requirement for a candidatespecies-common vaccine. At present, there are no studies that haveinvestigated the sequence conservation of the psaA gene amongpneumococcal types, specifically among encapsulated pneumococci whichcause the vast majority of serious disease. Therefore, a need exists toinvestigate the conservation of the gene in order to provide apolypeptide which can serve as a vaccine for multiple strains ofStreptococcus pneumoniae. The present invention fulfills that need byanalyzing psaA genes from the 23 serotypes in the 23-valentpolysaccharide vaccine and by providing a polypeptide and antibodies tothat polypeptide which are conserved among the S. pnuemoniae serotypesand which confer protection to Streptococcus pneumoniae infection.

SUMMARY OF THE INVENTION

[0010] In accordance with the purpose(s) of this invention, as embodiedand broadly described herein, this invention, in one aspect, relates toan isolated nucleic acid encoding the 37-kDa protein of Streptococcuspneumoniae as set forth in the Sequence Listing as SEQ ID NO:2. Theinvention also provides unique fragments of at least 10 nucleotides ofthe nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, whichcan be used in methods to detect the presence of Streptococcuspneumoniae in a sample and as immunogenic vaccines.

[0011] The invention further provides a purified polypeptide encoded bythe nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniaeas set forth in the Sequence Listing as SEQ ID NO:1, which can be usedas immunogenic vaccines.

[0012] In another aspect, the invention provides purified antibodieswhich bind to the 37-kDa protein of Streptococcus pneumoniae orfragments thereof. These antibodies can be used in methods to detect thepresence of Streptococcus pneumoniae in a sample and in therapeutic andprophylactic methods.

[0013] The advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

[0014] Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this application pertains.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included therein.

[0016] Before the present compounds and methods are disclosed anddescribed, it is to be understood that this invention is not limited tospecific proteins, specific methods, or specific nucleic acids, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

[0017] It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a nucleic acid” includes multiple copies of thenucleic acid and can also include more than one particular species ofnucleic acid molecule.

[0018] Nucleic Acids

[0019] In one aspect, the invention provides an isolated nucleic acidencoding the 37-kDa protein of Streptococcus pneumoniae as set forth inthe Sequence Listing as SEQ ID NO:2. The term “isolated” refers to anucleic acid which is essentially separated from other genes thatnaturally occur in S. pneumoniae. In one embodiment, the presentinvention provides an isolated nucleic acid encoding the 37-kDa proteinof Streptococcus pneumoniae wherein the nucleic acid is the nucleic acidset forth in the Sequence Listing as SEQ ID NO:1.

[0020] The nucleic acids of the present invention can include thepositive and/or negative RNA strand as well as the sense and/or nonsenseDNA strand, or any combinations thereof These nucleic acids include thegenomic DNA fragment encoding the 37-kDa protein and any subgenomicnucleic acids, including DNA and RNA, in the organism encoding all, or afragment of the 37-kDa protein. The nucleic acid can also be modified,such as nucleic acids containing methylated bases.

[0021] This nucleic acid can comprise the coding sequence for the 37-kDaprotein itself, or the coding sequence with the gene's upstream anddownstream regulatory sequences, or any combination thereof. Thisnucleic acid can, for example, comprise a DNA and include its ownpromoter, or another promoter can be operatively linked to the nucleicacid such that the coding sequence of the 37-kDa protein is expressed.Alternatively, the nucleic acid can comprise an RNA such that the RNA istranslated and the resulting polypeptide comprises the 37-kDa protein.Therefore sequences normally found associated with the 37-kDa proteincoding sequence, such as the promoter, and the translation signals, canbe substituted, deleted, or modified.

[0022] An isolated nucleic acid comprising a unique fragment of at least10 nucleotides of the nucleic acid set forth in the Sequence Listing asSEQ ID NO:1 is also provided. Unique fragments, as used herein means anucleic acid of at least 10 nucleotides that is not identical to anyother known nucleic acid sequence. Examples of the sequences of at least10 nucleotides that are unique to the nucleic acid set forth in theSequence Listing as SEQ ID NO: 1 can be readily ascertained by comparingthe sequence of the nucleic acid in question to sequences catalogued inGenBank, or any other sequence database, using computer programs such asDNASIS (Hitachi Engineering, Inc.) or Word Search or FASTA of theGenetics Computer Group (GCG) (Madison, Wis.), which search thecatalogued nucleotide sequences for similarities to the nucleic acid inquestion. If the sequence does not match any of the known sequences, itis unique. For example, the sequence of nucleotides 1-10 can be used tosearch the databases for an identical match. If no matches are found,then nucleotides 1-10 represent a unique fragment. Next, the sequence ofnucleotides 2-11 can be used to search the databases, then the sequenceof nucleotides 3-13, and so on up to nucleotides 1320 to 1330 of thesequence set forth in the Sequence Listing as SEQ ID NO:1. The same typeof search can be performed for sequences of 11 nucleotides, 12nucleotides, 13 nucleotides, etc. The possible fragments range from 10nucleotides in length to 1 nucleotide less than the sequence set forthin the Sequence Listing as SEQ ID NO: 1. These unique nucleic acids, aswell as degenerate nucleic acids can be used, for example, as primersfor amplifying nucleic acids from other strains of Streptococcuspneumoniae in order to isolate allelic variants of the 37-kDa protein,or as primers for reverse transcription of 37-kDa protein RNA, or asprobes for use in detection techniques such as nucleic acidhybridization. One skilled in the art will appreciate that even though anucleic acid of at least 10 nucleotides is unique to a specific gene,that nucleic acid fragment can still hybridize to many other nucleicacids and therefore be used in techniques such as amplification andnucleic acid detection.

[0023] Also provided are nucleic acids which encode allelic variants ofthe 37-kDa protein of S. pneumoniae set forth in the Sequence Listing asSEQ ID NO:2, and those proteins. As used herein, the term “allelicvariations” or “allelic variants” is used to describe the same, orsimilar 37-kDa pneumococcal surface adhesin proteins that are divergedfrom the 37-kDa Streptococcus pneumoniae protein set forth in theSequence Listing as SEQ ID NO:2 by less than 15% in their correspondingamino acid identity. In another embodiment, these allelic variants areless than 10% divergent in their corresponding amino acid identity. Inanother embodiment, these allelic variants are less than 7% divergent intheir corresponding amino acid identity. In another embodiment, theseallelic variants are less than 5% divergent in their corresponding aminoacid identity. In another embodiment, these allelic variants are lessthan 3% divergent in their corresponding amino acid identity. In anotherembodiment, these allelic variants are less than 2% divergent in theircorresponding amino acid identity. In yet another embodiment, theseallelic variants are less than 1% divergent in their corresponding aminoacid identity. These amino acids can be substitutions within the aminoacid sequence set forth in the Sequence Listing as SEQ ID NO:2, they canbe deletions from the amino acid sequence set forth in the SequenceListing as SEQ ID NO:2, and they can be additions to the amino acidsequence set forth in the Sequence Listing as SEQ ID NO:2.

[0024] The homology between the protein coding region of the nucleicacid encoding the allelic variant of the 37-kDa protein is preferablyless than 20% divergent from the region of the nucleic acid set forth inthe Sequence Listing as SEQ ID NO:1 encoding the 37-kDa protein. Inanother embodiment, the corresponding nucleic acids are less than 15%divergent in their sequence identity. In another embodiment, thecorresponding nucleic acids are less than 10% divergent in theirsequence identity In another embodiment, the corresponding nucleic acidsare less than 7% divergent in their sequence identity. In anotherembodiment, the corresponding nucleic acids are less than 5% divergentin their sequence identity In another embodiment, the correspondingnucleic acids are less than 4% divergent in their sequence identity. Inanother embodiment, the corresponding nucleic acids are less than 3%divergent in their sequence identity. In another embodiment, thecorresponding nucleic acids are less than 2% divergent in their sequenceidentity. In yet another embodiment, the corresponding nucleic acids areless than 1% divergent in their sequence identity. In particular, thenucleic acid variations can create up to about 15% amino acid sequencevariation from the protein set forth in the Sequence Listing as SEQ IDNO:2.

[0025] One skilled in the art will appreciate that nucleic acidsencoding homologs or allelic variants of the 37-kDa protein set forth inthe Sequence Listing as SEQ ID NO:2 can be isolated from relatedgram-positive bacteria in a manner similar to that used to isolate thenucleic acid set forth in the Sequence Listing of the present inventionas SEQ ID NO: 1. For example, given the sequence of the primers used toamplify the nucleic acid set forth in the sequence listing as SEQ ID NO:1, one can use these or similar primers to amplify a homologous genefrom related gram-positive bacteria.

[0026] Alternatively, allelic variants can be identified and isolated bynucleic acid hybridization techniques. Probes selective to the nucleicacid set forth in the Sequence Listing as SEQ ID NO:1 can be synthesizedand used to probe nucleic acid from the various serotypes of S.pneumoniae. High sequence complementarity and stringent hybridizationconditions can be selected such that the probe selectively hybridizes toallelic variants of the sequence set forth in the Sequence Listing asSEQ ID NO:1. For example, the selectively hybridizing nucleic acids ofthe invention can have at least 70%, 80%, 85%, 90% 95%, 97%, 98% and 99%complementarity with the segment of the sequence to which it hybridizes.The nucleic acids can be at least 10, 12, 50, 100, 150, 200, 300, 500750, or 1000 nucleotides in length. Thus, the nucleic acid can be acoding sequence for the 37-kDa protein or fragments thereof that can beused as a probe or primer for detecting the presence of M. tuberculosis.If used as primers, the invention provides compositions including atleast two nucleic acids which hybridize with different regions so as toamplify a desired region. Depending on the length of the probe orprnmer, target region can range between 70% complementary bases and fullcomplementanty and still hybridize under stringent conditions. Forexample, for the purpose of diagnosing the presence of an allelicvariant of the sequence set forth in the Sequence Listing as SEQ ID NO:1the degree of complementarity between the hybridizing nucleic acid(probe or primer) and the sequence to which it hybridizes is at leastenough to distinguish hybridization with a nucleic acid from unrelatedbacteria. The invention provides examples of nucleic acids unique to SEQID NO: 1 in the Sequence Listing so that the degree of complementarityrequired to distinguish selectively hybridizing from nonselectivelyhybridizing nucleic acids under stringent conditions can be clearlydetermined for each nucleic acid. One skilled in the art will appreciatethat sequences can be added to either one end or both ends of uniquefragments, for example, to aid subsequent cloning, expression, ordetection of the fragment.

[0027] “Stringent conditions” refers to the washing conditions used in ahybridization protocol. In general, the washing conditions should be acombination of temperature and salt concentration chosen so that thedenaturation temperature is approximately 5-20° C. below the calculatedT_(m) of the nucleic acid hybrid under study. The temperature and saltconditions are readily determined empirically in preliminary experimentsin which samples of reference DNA immobilized on filters are hybridizedto the probe or protein coding nucleic acid of interest and then washedunder conditions of different stringencies. The T_(m) of such anoligonucleotide can be estimated by allowing 2° C. for each A or Tnucleotide, and 4° C. for each G or C. For example, an 18 nucleotideprobe of 50% G+C would, therefore, have an approximate T_(m) of 54° C.

[0028] In another aspect, the present invention provides an isolatednucleic acid comprising the nucleic acid as set forth in the SequenceListing as SEQ ID NO:3.

[0029] In another aspect, the present invention provides an isolatednucleic acid comprising the nucleic acid as set forth in the SequenceListing as SEQ ID NO:4.

[0030] The nucleic acid encoding a 37-kDa protein may be obtained by anynumber of techniques known to one skilled in the art. One method is tosynthesize a recombinant nucleic acid molecule. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein or regulatory regionare readily obtainable through automated DNA synthesis. A nucleic acidfor one strand of a double-stranded molecule can be synthesized andhybridized to its complementary strand. One can design theseoligonucleotides such that the resulting double-stranded molecule haseither internal restriction sites or appropriate 5′ or 3′ overhangs atthe termini for cloning into an appropriate vector. Double-strandedmolecules coding for relatively large proteins or regulatory regions canbe synthesized by first constructing several different double-strandedmolecules that code for particular regions of the protein or regulatoryregion, followed by ligating these DNA molecules together. For example,Cunningham et al. (“Receptor and Antibody Epitopes in Human GrowthHormone Identified by Homolog-Scanning Mutagenesis,” Science,243:1330-1336 (1989)), have constructed a synthetic gene encoding thehuman growth hormone gene by first constructing overlapping andcomplementary synthetic oligonucleotides and ligating these fragmentstogether. See also, Ferretti, et al. (Proc. Nat. Acad. Sci. 82:599-603(1986)), wherein synthesis of a 1057 base pair synthetic bovinerhodopsin gene from synthetic oligonucleotides is disclosed. Once theappropriate DNA molecule is synthesized, this DNA can be cloneddownstream of a promoter. Techniques such as this are routine in the artand are well documented.

[0031] An example of another method of obtaining a nucleic acid encodinga 37-kDa surface adhesin A protein is to isolate that nucleic acid fromthe organism in which it is found and clone it in an appropriate vector.For example, a DNA or cDNA library can be constructed and screened forthe presence of the nucleic acid of interest. The probe used to screenthe library can be designed to be selective for the 6B serotype protein.Methods of constructing and screening such libraries are well known inthe art and kits for performing the construction and screening steps arecommercially available (for example, Stratagene Cloning Systems, LaJolla, Calif.). Once isolated, the nucleic acid can be directly clonedinto an appropriate vector, or if necessary, be modified to facilitatethe subsequent cloning steps. Such modification steps are routine, anexample of which is the addition of oligonucleotide linkers whichcontain restriction sites to the termini of the nucleic acid. Generalmethods are set forth in Sambrook et al., “Molecular Cloning, aLaboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

[0032] Yet another example of a method of obtaining a Streptococcal37-kDa surface adhesin A encoding nucleic acid is to amplify the nucleicacid from the nucleic acids found within the host organism.Amplification procedures are well known to those skilled in the art, forexample see Innis et al. “PCR Protocols: A Guide to Methods andApplications” Academic Press, Inc. 1990. An example of amplification ofa nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniaeserotype 6B is discussed in the Example contained herein.

[0033] 37-kDa Protein

[0034] The present invention also provides a purified polypeptide as setforth in the Sequence Listing a SEQ ID NO:2 and a purified polypeptideencoded by a nucleic acid comprising a unique fragment of at least 10nucleotides of SEQ ID NO:1. The protein can be used as a vaccinecomponent as well as a reagent for identifying host antibodies raisedagainst Streptococcus pneumoniae during infection. The purified proteincan also be used in methods for detecting the presence of Streptococcuspneumoniae.

[0035] Unique fragments of the 37-kDa protein can be identified in thesame manner as that used to identify unique nucleic acids. For example,a sequence of 3 amino acids or more, derived from the sequence of the37-kDa protein as set forth in the Sequence Listing as SEQ ID NO:2 canbe used to search the protein sequence databases. Those that do notmatch a known sequence are therefore unique.

[0036] “Purified protein” as used herein means the protein or fragmentis sufficiently free of contaminants or cell components with which theprotein normally occurs to distinguish the protein from the contaminantsor cell components. It is not contemplated that “purified” necessitateshaving a preparation that is technically totally pure (homogeneous), butpurified as used herein means the protein or polypeptide fragment issufficiently separated from contaminants or cell components with whichit normally occurs to provide the protein in a state where it can beused in an assay, such as immunoprecipitation or ELISA. For example, the“purified” protein can be in an electrophoretic gel.

[0037] Once a nucleic acid encoding a 37-kDa pneumococcal surfaceadhesin protein of serotype 6B, or a fragment of that nucleic acid, isconstructed, modified, or isolated, that nucleic acid can then be clonedinto an appropriate vector, which can direct the in vivo or in vitrosynthesis of that 37-kDa pneumococcal surface adhesin protein, orfragment thereof. The vector is contemplated to have the necessaryfunctional elements that direct and regulate transcription of theinserted gene, or gene fragment. These functional elements include, butare not limited to, a promoter, regions upstream or downstream of thepromoter, such as enhancers that may regulate the transcriptionalactivity of the promoter, an origin of replication, appropriaterestriction sites to facilitate cloning of inserts adjacent to thepromoter, antibiotic resistance genes or other markers which can serveto select for cells containing the vector or the vector containing theinsert, RNA splice junctions, a transcription termination region, or anyother region which may serve to facilitate the expression of theinserted gene or gene fragment. (See generally, Sambrook et al.).

[0038] There are numerous E. coli (Escherichia coli) expression vectorsknown to one of ordinary skill in the art which are useful for theexpression of the nucleic acid insert. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, apromoter system from phage lambda, or other phage promoters such as T4or T7 promoters. The promoters will typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences for example, for initiating and completing transcription andtranslation. If necessary, an amino terminal methionine can be providedby insertion of a Met codon 5′ and in-frame with the downstream nucleicacid insert. Also, the carboxy-terminal extension of the nucleic acidinsert can be removed using standard oligonucleotide mutagenesisprocedures.

[0039] Alternatively, viral expression systems can be used to expressthe nucleic acid of the present invention, or fragments thereof. Forexample, vaccinia virus vectors can accept large inserts and can be usedto express foreign genes for vaccination purposes. (See, e.g., Friedman,T. Science 244:1275 (1989)). Other viral expression systems, such as thebaculovirus expression system, are also commonly used in the art.

[0040] Additionally, yeast expression can be used. There are severaladvantages to yeast expression systems. First, evidence exists thatproteins produced in a yeast secretion systems exhibit correct disulfidepairing. Second, post-translational glycosylation is efficiently carriedout by yeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake et al.,“∝-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae.” Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems.

[0041] Mammalian cells permit the expression of proteins in anenvironment that favors important post-translational modifications suchas folding and cysteine pairing, addition of complex carbohydratestructures, addition of lipid moieties, and secretion of active protein.Vectors useful for the expression of active proteins in mammalian cellsare characterized by insertion of the protein coding sequence between astrong viral promoter and a polyadenylation signal. The vectors cancontain genes conferring hygromycin resistance, gentamicin resistance,or other genes or phenotypes suitable for use as selectable markers, ormethotrexate resistance for gene amplification. The chimeric proteincoding sequence can be introduced into a Chinese hamster ovary (CHO)cell line using a methotrexate resistance-encoding vector, or other celllines using suitable selection markers. Presence of the vector DNA intransformed cells can be confirmed by Southern blot analysis. Productionof RNA corresponding to the insert coding sequence can be confirmed byNorthern blot analysis. A number of other suitable host cell linescapable of secreting intact human proteins have been developed in theart, and include the CHO cell lines, HeLa cells, myeloma cell lines,Jurkat cells, etc. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer, and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation, transduction, andelectroporation are commonly utilized for prokaryotic cells, whereascalcium phosphate, DEAE dextran, or lipofectin mediated transfection orelectroporation may be used for other cellular hosts.

[0042] Alternative vectors for the expression of genes in mammaliancells, those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VIII,hepatitis B virus surface antigen, protease Nexinl, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

[0043] Expression of the gene or hybrid gene can be by either in vivo orin vitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector.Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of mRNA. In such in vitro transcription systems, thenucleic acid encoding the 37-kDa pneumococcal surface adhesin proteinwould be cloned into an expression vector adjacent to a transcriptionpromoter. For example, the Bluescript II cloning and expression vectorscontain multiple cloning sites which are flanked by strong prokaryotictranscription promoters. (Stratagene Cloning Systems, La Jolla, Calif.).Kits are available which contain all the necessary reagents for in vitrosynthesis of an RNA from a DNA template such as the Bluescript vectors.(Stratagene Cloning Systems, La Jolla, Calif.). RNA produced in vitro bya system such as this can then be translated in vitro to produce thedesired 37-kDa pneumococcal surface adhesin protein. (Stratagene CloningSystems, La Jolla, Calif.).

[0044] Another method of producing a 37-kDa pneumococcal surface adhesinprotein is to link two peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to a 37-kDa pneumococcal surfaceadhesin protein can be synthesized by standard chemical reactions,either continuous synthesis or step-wise synthesis. For example, apartial polypeptide can be synthesized and not cleaved from itssynthesis resin whereas another fragment can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform a 37-kDa pneumococcal surface adhesin protein. (Grant G. A.,“Synthetic Peptides: A User Guide,” W.H. Freeman and Co., N.Y. (1992)and Bodansky, M. and Trost, B., Ed., “Principles of Peptide Synthesis,”Springer-Verlag Inc., N.Y. (1993)). Alternatively, the 37-kDapneumococcal surface adhesin protein can by independently synthesized mvivo as described above. Once isolated, these independent polypeptidesmay be linked to form a 37-kDa pneumococcal surface adhesin protein viasimilar peptide condensation reactions.

[0045] For example, enzymatic ligation of cloned or synthetic peptidesegments can allow relatively short peptide fragments to be joined toproduce larger peptide fragments, polypeptides or whole protein domains(Abrahmsen et al., Biochemistry, 30:4151 (1991)). Alternatively, nativechemical ligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.,“Synthesis of Proteins by Native Chemical Ligation,” Science,266:776-779 (1994)). The first step is the chemoselective reaction of anunprotected synthetic peptide-∝-thioester with another unprotectedpeptide segment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis et al., FEBSLett., 307:97 (1987), Clark-Lewis et al., J. Biol. Chem., 269:16075(1994), Clark-Lewis et al, Biochem. 30:3128 (1991), and Rajarathnam etal., Biochem. 29:1689 (1994)).

[0046] Alternatively, unprotected peptide segments can be chemicallylinked where the bond formed between the peptide segments as a result ofthe chemical ligation is an unnatural (non-peptide) bond (Schnolzer etal., Science, 256:221 (1992)). This technique has been used tosynthesize analogs of protein domains as well as large amounts ofrelatively pure proteins with full biological activity (deLisle Miltonet al., “Techniques in Protein Chemistry IV,” Academic Press, New York,pp. 257-267 (1992)).

[0047] The invention also provides fragments of the 37-kDa pneumococcalsurface adhesin protein. The polypeptide fragments of the presentinvention can be recombinant proteins obtained by cloning nucleic acidsencoding fragments of the polypeptide in an expression system capable ofproducing the polypeptide fragments thereof, as described above for the37-kDa protein. For example, one can determine an immunoreactive regionof a 37-kDa pneumococcal surface adhesin protein which can cause asignificant immune response, clone the nucleic acid encoding thatpolypeptide into an expression vector, and isolate that particularpolypeptide for further uses, such as diagnostics, therapy, andvaccination. In one example, amino acids found to not contribute to theimmunoreactivity and/or specificity can be deleted without a loss in therespective activity.

[0048] For example, amino or carboxy-terminal amino acids, can besequentially removed from the 37-kDa pneumococcal surface adhesinprotein and the immunoreactivity tested in one of many available assays.Alternatively, internal amino acids can be sequentially removed and theimmunoreactivity tested for each of the deletions. In another example, afragment of a 37-kDa pneumococcal surface adhesin protein can comprise amodified polypeptide wherein at least one amino acid has beensubstituted for the naturally occurring amino acid at specificpositions, or a portion of either amino terminal or carboxy terminalamino acids, or even an internal region of the polypeptide, can bereplaced with a polypeptide fragment or other moiety, such as biotin,which can facilitate in the purification of the modified 37-kDapneumococcal surface adhesin protein. For example, a modified 37-kDapneumococcal surface adhesin protein can be fused to a maltose bindingprotein, through either peptide chemistry of cloning the respectivenucleic acids encoding the two polypeptide fragments into an expressionvector such that the expression of the coding region results in a hybridpolypeptide. The hybrid polypeptide can be affinity purified by passingit over an amylose affinity column, and the modified 37-kDa pneumococcalsurface adhesin protein can then be separated from the maltose bindingregion by cleaving the hybrid polypeptide with the specific proteasefactor Xa. (See, e.g., New England Biolabs Product Catalog, 1996, pg.164.)

[0049] Immunoreactive fragments of a 37-kDa pneumococcal surface adhesinprotein can also be synthesized directly or obtained by chemical ormechanical disruption of larger 37-kDa pneumococcal surface adhesinprotein. An immunoreactive fragment is defined as an amino acid sequenceof at least about 6 consecutive amino acids derived from the naturallyoccurring amino acid sequence, which has the relevant activity, e g.,evoking an immune response.

[0050] The fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the immunoreactivity of the peptide is not significantlyimpaired compared to the 37-kDa pneumococcal surface adhesin protein.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, etc. In any case, the peptide must possess a bioactiveproperty, such as immunoreactivity. Functional or active regions of the37-kDa pneumococcal surface adhesin protein may be identified bymutagenesis of a specific region of the protein, followed by expressionand testing of the expressed polypeptide. Such methods are readilyapparent to a skilled practitioner in the art and can includesite-specific mutagenesis of the nucleic acid encoding the receptor.(See, e.g., Smith, M. “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462(1985) and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605-610 (1991)).

[0051] Antibodies

[0052] The present invention also provides a purified antibody whichselectively binds with the polypeptide encoded by the nucleic acid setforth in the sequence listing as SEQ ID NO: 1, or a polypeptide encodedby a unique fragment of at least 10 nucleotides of SEQ ID NO: 1. Theantibody (either polyclonal or monoclonal) can be raised to the 37-kDapneumococcal surface adhesin protein of a unique fragment thereof, inits naturally occurring form and in its recombinant form. The antibodycan be used in techniques or procedures such as diagnostics, treatment,or vaccination.

[0053] Antibodies can be made by many well-known methods (See, e.g.Harlow and Lane, “Antibodies; A Laboratory Manual” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigencan be injected into an animal in an amount and in intervals sufficientto elicit an immune response. Antibodies can either be purifieddirectly, or spleen cells can be obtained from the animal. The cells canthen fused with an immortal cell line and screened for antibodysecretion. The antibodies can be used to screen nucleic acid clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced. (See, for example, Kelly et al., Bio/Technology,10:163-167 (1992); Bebbington et al., Bio/Technology, 10:169-175(1992)).

[0054] The phrase “selectively binds” with the polypeptide refers to abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies boundto a particular protein do not bind in a significant amount to otherproteins present in the sample. Selective binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select antibodies selectively bind with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies selectively immunoreactive with a protein. SeeHarlow and Lane “Antibodies, A Laboratory Manual” Cold Spring HarborPublications, New York, (1988), for a description of immunoassay formatsand conditions that could be used to determine selective binding.

[0055] In some instances, it is desirable to prepare monoclonalantibodies from various hosts. A description of techniques for preparingsuch monoclonal antibodies may be found in Stiles et al., editors,“Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos,Calif., Fourth Edition) and references cited therein, and in Harlow andLane (“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York, (1988)).

[0056] The present invention also provides a monoclonal antibodydesignated 1E7A3D7C2, or a fragment thereof which retains thecharacteristics of antibody 1E7A3D7C2, such as its binding specificityand its binding affinity.

[0057] The present invention also provides a monoclonal antibodydesignated 1B6E12H9, or a fragment thereof which retains thecharacteristics of antibody 1B6E12H9.

[0058] The present invention also provides a monoclonal antibodydesignated 3C4D5C7, or a fragment thereof which retains thecharacteristics of antibody 3C4D5C7.

[0059] The present invention also provides a monoclonal antibodydesignated 4E9G9D3, or a fragment thereof which retains thecharacteristics of antibody 4E9G9D3.

[0060] The present invention also provides a monoclonal antibodydesignated 4H5C10F3, or a fragment thereof which retains thecharacteristics of antibody 4H5C10F3.

[0061] The present invention also provides a monoclonal antibodydesignated 6F6F9C8, or a fragment thereof which retains thecharacteristics of antibody 6F6F9C8.

[0062] The present invention also provides a monoclonal antibodydesignated 8G12G11B10, or a fragment thereof which retains thecharacteristics of antibody 8G12G11B10.

[0063] Vaccines

[0064] Also provided by the present invention is a vaccine comprising animmunogenic polypeptide encoded by the nucleic acid as set forth in theSequence Listing as SEQ ID NO: 1, or a unique fragment of at least 10nucleotides of SEQ ID NO: 1. The polypeptides provided by the presentinvention can be used to vaccinate a subject for protection from aparticular disease, infection, or condition caused by the organism fromwhich the 37-kDa pneumococcal surface adhesin protein of a uniquefragment thereof was derived.

[0065] Polypeptides of a 37-kDa pneumococcal surface adhesin protein ofserotype 6B or a unique fragment thereof, therefore, can be used toinoculate a host organism such that the host generates an active immuneresponse to the presence of the polypeptide or polypeptide fragmentwhich can later protect the host from infection by organism from whichthe polypeptide was derived. One skilled in the art will appreciate thatan immune response, especially a cell-mediated immune response, to a37-kDa pneumococcal surface adhesin protein from a specific strain canprovide later protection from reinfection or from infection from aclosely related strain. The 37-kDa protein provided by the presentinvention, however, is relatively conserved among many of the variousserotypes of S. pneumoniae and can serve as a multivalent vaccine.

[0066] Immunization with the 37-kDa pneumococcal surface adhesin proteincan be achieved through artificial vaccination. (Kuby, J. “Immunology”W. H. Freeman and Co. New York, 1992). This immunization may be achievedby administering to subjects the 37-kDa pneumococcal surface adhesinprotein either alone or with a pharmaceutically acceptable carrier.

[0067] Immunogenic amounts of the 37-kDa pneumococcal surface adhesinprotein can be determined using standard procedures. Briefly, variousconcentrations of the present polypeptide are prepared, administered tosubjects, and the immunogenic response (e.g., the production ofantibodies to the polypeptide or cell mediated immunity) to eachconcentration is determined. Techniques for monitoring the immunogenicresponse, both cellular and humoral, of patients after inoculation withthe polypeptide, are very well known in the art. For example, samplescan be assayed using enzyme-linked immunosorbent assays (ELISA) todetect the presence of specific antibodies, such as serum IgA (Hjelt etal. J. Med. Virol. 21:39-47, (1987)), or lymphocyte or cytokineproduction can be monitored. The specificity of a putative immunogenicantigen of any particular polypeptide can be ascertained by testingsera, other fluids or lymphocytes from the inoculated patient forcross-reactivity with other closely related 37-kDa pneumococcal surfaceadhesin proteins.

[0068] The amount of a polypeptide of the 37-kDa pneumococcal surfaceadhesin protein administered will depend on the subject, the conditionof the subject, the size of the subject, etc., but will be at least animmunogenic amount. The polypeptide can be formulated with adjuvants andwith additional compounds, including cytokines, with a pharmaceuticallyacceptable carrier.

[0069] It is also contemplated that immunization against Streptococcuspneumoniae can be achieved by a “naked” DNA vaccine approach. Briefly,DNA constructs containing promoter sequences upstream of the 37-kDaprotein or specific antigen coding sequences can be injected into muscletissue or administered via the mucosa and result in expression of viralantigens that induce a protective immune response.

[0070] The pharmaceutically acceptable carrier or adjuvant in thevaccine of the present invention can be selected by standard criteria(Arnon, R Ed.) “Synthetic Vaccines” I:83-92, CRC Press, Inc. Boca Raton,Fla., 1987). By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected compound withoutcausing any undesirable biological effects or interacting in aundesirable manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier oradjuvant may depend on the method of administration and the particularpatient.

[0071] Methods of administration can be by oral, sublingual mucosal,inhaled, absorbed, or by injection. Actual methods of preparing theappropriate dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences (Martin, E. W. (ed.) latest edition Mack Publishing Co.,Easton, Pa.

[0072] Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

[0073] Detection Methods

[0074] The present invention also provides a method of detecting thepresence of the Streptococcus pneumoniae in a sample, comprising thesteps of contacting a sample suspected of containing Streptococcuspneumoniae with nucleic acid primers capable of hybridizing to a nucleicacid comprising a unique portion of the nucleic acid set forth in theSequencing Listing as SEQ ID NO:1, amplifying the nucleic acid,detecting the presence of an amplification product, the presence of theamplification product indicating the presence of Streptococcuspneumoniae in the sample. Alternatively, a unique fragment of thenucleic acid of SEQ ID NO: 1 can be used to specifically identify anon-selectively amplified nucleic acid.

[0075] The specific amplification methods are well known in the art. Forexample, and as disclosed in the Example contained herein the polymerasechain reaction (PCR) can be used to amplify nucleic acid in a samplespecific for Streptococcus pneumoniae. Other amplification techniquescan also be used to detect the presence of Streptococcus pneumoniae in asample, such as the ligase chain reaction (LCR), the self-sustainedsequence replication (3SR) system, the transcription-based amplificationsystem (TAS), and the RNA replication system based on Qβ replicase.

[0076] The amplified nucleic acid can be detected in any number ofdetection assays. For example, the primers can be radio-labeled suchthat the amplification product containing these primers can be detectedby the detecting the radioactive decay from those primers.Alternatively, the primers can contain other detectable moieties, suchas biotin, or the amplified nucleic acid can be stained and visualized,such as with ethidium bromide staining.

[0077] The present invention also provides a method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof contacting an antibody-containing sample from the subject withpurified polypeptide encoded by the nucleic acid set forth in theSequence Listing as SEQ ID NO: 1, or a purified polypeptide encoded by anucleic acid comprising a unique fragment of at least 10 nucleotides ofSEQ ID NO: 1, and detecting the binding of the antibody with thepolypeptide, the binding indicating the presence of Streptococcuspneumoniae in the subject.

[0078] The present invention further provides a method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof contacting a sample from the subject with an antibody whichselectively binds the purified polypeptide encoded by the nucleic acidset forth in the Sequence Listing as SEQ ID NO: 1, or a purifiedpolypeptide encoded by a nucleic acid comprising a unique fragment of atleast 10 nucleotides of SEQ ID NO: 1 and detecting the binding of theantibody with an antigen, the binding indicating the presence ofStreptococcus pneumoniae in the subject.

[0079] There are numerous immunodiagnostic methods that can be used todetects antigen or antibody as the following non-inclusive examplesillustrate. These methods, as well as others, can not only detect thepresence of antigen or antibody, but quantitate antigen or antibody aswell.

[0080] Immunoassays such as immunofluorescence assays (IFA), enzymelinked immunosorbent assays (ELISA) and immunoblotting can be readilyadapted to accomplish the detection and quantitation of the antigen orantibody. An ELISA method effective for the detection of the antigen,for example, can be as follows: (1) bind the antibody to a substrate;(2) contact the bound antibody with a fluid or tissue samplecontaining:the antigen; (3) contact the above with a secondary antibodybound to a detectable moiety (e.g., horseradish peroxidase enzyme oralkaline phosphatase enzyme); (4) contact the above with the substratefor the enzyme; (5) contact the above with a color reagent; (6) observecolor change. The above method can be readily modified to detectantibody as well as antigen.

[0081] Another immunologic technique that can be useful in the detectionutilizes monoclonal antibodies (MAbs) for detection of antibodies thatspecifically bind a specific antigen. Briefly, sera or other body fluidfrom the subject is reacted with the antigen bound to a substrate (e.g.an ELISA 96-well plate). Excess sera is thoroughly washed away. Alabeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonalantibody is then reacted with the previously reacted antigen-serumantibody complex. The amount of inhibition of monoclonal antibodybinding is measured relative to a control (no patient serum antibody).The degree of monoclonal antibody inhibition can be a specific test fora particular species or subspecies or variety or strain since it isbased on monoclonal antibody binding specificity. MAbs can also be usedfor detection directly in cells by IFA.

[0082] A micro-agglutination test can also be used to detect thepresence of antibodies in a subject. Briefly, latex beads (or red bloodcells) are coated with the antigen and mixed with a sample from thesubject, such that antibodies in the tissue or body fluids that arespecifically reactive with the antigen crosslink with the antigen,causing agglutination. The agglutinated antigen-antibody complexes forma precipitate, visible with the naked eye or detectable by aspectrophotometer. In a modification of the above test, antibodiesspecifically reactive with the antigen can be bound to the beads andantigen in the tissue or body fluid thereby detected.

[0083] In addition, as in a typical sandwich assay, the antibody can bebound to a substrate and contacted with the antigen. Thereafter, alabeled secondary antibody is bound to epitopes not recognized by thefirst antibody and the secondary antibody is detected.

[0084] In the diagnostic methods taught herein, the antigen can be boundto a substrate and contacted by a fluid sample such as serum, urine,saliva or gastric juice. This sample can be taken directly from thesubject or in a partially purified form. In this manner, antibodiesspecific for the antigen (the primary antibody) will specifically bindwith the bound antigen. Thereafter, a secondary antibody bound to, orlabeled with, a detectable moiety can be added to enhance the detectionof the primary antibody. Generally, the secondary antibody or otherligand which binds specifically with a different epitope of the antigenor nonspecifically with the ligand or bound antibody, will be selectedfor its ability to bind with multiple sites on the primary antibody.Thus, for example, several molecules of the secondary antibody can bindwith each primary antibody, making the primary antibody more detectable.

[0085] The detectable moiety will allow visual detection of aprecipitate or a color change, visual detection by microscopy, orautomated detection by spectrometry, radiometric measurement or thelike. Examples of detectable moieties include fluorescein and rhodamine(for fluorescence microscopy), horseradish peroxidase (for either lightor electron microscopy and biochemical detection), biotin-streptavidin(for light or electron microscopy) and alkaline phosphatase (forbiochemical detection by color change). The detection methods andmoieties used can be selected, for example, from the list above or othersuitable examples by the standard criteria applied to such selections(Harlow et al., “Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)).

[0086] Methods of Treating and Preventing Infection

[0087] The present invention also provides a method of preventingStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a prophylactically effective amount of avaccine comprising an immunogenic polypeptide encoded by the nucleicacid encoding the 37-kDa protein of Streptococcus pneumoniae as setforth in the Sequence Listing as SEQ ID NO: 1, or an immunogenicpolypeptide encoded by a nucleic acid comprising a unique fragment of atleast 10 nucleotides of SEQ ID NO:1., either alone or with apharmaceutically acceptable carrier.

[0088] The present invention further provides a method of preventingStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a prophylactically effective amount of ananti-idiotype antibody to the polypeptide encoded by the nucleic acid asset forth in the Sequence Listing as SEQ ID NO: 1, or a polypeptideencoded by a nucleic acid comprising a unique fragment of at least 10nucleotides of SEQ ID NO: 1, either alone or with a pharmaceuticallyacceptable carrier.

[0089] Anti-idiotype antibodies represent the image of the originalantigen and can serve as a vaccine to induce an immune response to apathogenic antigen, therefore avoiding immunization with the pathogenitself. This type of protection has been demonstrated by immunizing micewith anti-idiotype antibody to the binding site of TEPC-15, the majorcomponent of the pneumococcal cell wall C polysaccharide. Mice immunizedwith these anti-idiotype antibodies were immune when they were laterchallenged with live pneumococci. Mice have also been used todemonstrate anti-idiotype antibodies can provide protection againsthepatitis B virus, rabies virus, Sendai virus, Streptococcus pneumoniae,Listeria monocytogenes, Trypanosoma rhodesiense, and Schistosomamansoni. (See, Kuby, J. “Immunology” W. H. Freeman and Co. New York,1992).

[0090] The present invention further provides a method of treating aStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a therapeutically effective amount of anantibody to the polypeptide encoded by the nucleic acid as set forth inthe Sequence Listing as SEQ ID NO: 1, or a polypeptide encoded by anucleic acid comprising a unique fragment of at least 10 nucleotides ofSEQ ID NO: 1, either alone or with a pharmaceutically acceptablecarrier.

[0091] Treating a subject already infected with a particular organism byadministering to the subject antibody against the organism is well knownin the art. For example, immune globulin isolated from animals or humanspreviously exposed to rabies virus is currently a therapy for rabiesvirus infection. Better treatment of infected individuals can beachieved by administering to those individuals monoclonal antibodiessince those monoclonals react or bind more specifically that thepolyclonals. (See, e.g. Kaplan et al. “Rabies” Sci. Am. 242:120-134(1980)).

[0092] The following example is put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the attenuated prokaryotes claimed herein are made and evaluated,and demonstrates the methods of the present invention, and is intendedto be purely exemplary of the invention and is not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. and pressure is at or near atmospheric.

EXAMPLES

[0093] Bacterial strains The S. pneumoniae strain R36A was kindlyprovided by D. E. Briles (University of Alabama at Birmingham).Twenty-four serotypes of S. pneumoniae were provided by R. Facklam,Centers for Disease Control (CDC), Atlanta, Ga. These serotypes are 1,2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11F, 11A, 12F, 14, 15B, 18C,19A, 19F; 20, 22F, 23F, and 33F. Entex, ococcus avium, E. casselifavus,and E. gallinarum were also provided by R. Facklam. Anaerobic bacteriawere obtained from V. R. Dowell, CDC. These included Bacteroidesasaccharolyticus, B. fragilis, B. intermedius, B. thetaiotaomicron,Eubacterium lentum, Fusobacterium necrophorum, F. nucleatum,Peptostreptococcus anaerobius, P. asaccharolyticus, Propionibacteriumacnes, and Staphylococcus saccharolyticus. Branhamella catarrhalis andBordetella parapertussis were obtained from R. Weaver, CDC.Mycobacterium tuberculosis was provided by R. C. Good, CDC. R. Barnes,CDC, provided Chlamydia pneumoniae. The following remaining bacteriawere from the stock collection of the Immunology Laboratory, CDC:Bordetella pertussis, Enterobacter aerogenes, E. agglomerans, E.cloacae, E. gergoviae, Escherichia coli, Klebsiella pneumoniae,Haemophilus influenzae (types a-f), Legionella micdadei, L. pneumophila,Mycoplasma pneumoniae, Pseudomonas aeruginosa, Serratia marcescens,Staphylococcus aureus, Streptococcus agalactiae, S. equisimilis, S.pyogenes, and group G streptococci.

[0094] Production of MAbs Female BALB/c mice were immunized with wholecell suspensions of S. pneumoniae R36A, a rough derivative of thecapsular type 2 strain D39 (Avery et al. (1944) J. Exp. Med.79:137-157). The mice were immunized by intravenous injection threetimes and intraperitoneal injection one time. The maximum number ofcells injected at any time was 10⁸. Fusion was done on day 25 by usingstandard procedures (Clafin et al. (1978) Curr. Top. Microbiol. Immunol.81:107-109). Spleen cells of 4 mice were fused with Sp2/0-Ag14 myelomacells (Schulman et al. (1978) Nature (London) 276:269-270). Culturefluids of the growing hybridomas were tested for antibodies to S.pneumoniae whole cells in an ELISA. A clone designated 1E7A3D7C2 was oneof 10 selected for further study.

[0095] ELISA Screening of hybridoma culture supernatants was done byELISA. U-bottom microtitration plates (Costar, Cambridge, Mass.) weresensitized with 50 μl of S. pneumoniae whole cell suspension (10⁹CFU/ml) diluted 1:4,000 in 0.1 M carbonate buffer, pH 9.6, and kept for16 h at 4° C. The plates were washed 5 times with 0.9% NaCl containing0.05% Tween 20,NaCl-T). Culture supernatants (50 μl) from the fusionplates were added to 50 μl of a solution containing 2% bovine serumalbumin (BSA), 10% normal rabbit serum, 0.3% Tween-20, and 0.02%Merthiolate in phosphate buffered saline (PBS), pH 7.2, (ELISA diluent)(Wells. et al. (1987) J. Clin. Microbiol. 25:516-521) in the plates andwere incubated for 30 min at 37° C. The plates were washed 5 times withNaCl-T. Fifty microliters of goat anti-mouse immunoglobulin horseradishperoxidase conjugate diluted in ELISA diluent was added to each well.The plates were incubated for 30 min at 370C. The plates were washed,and 50 μl of 3,3′,5,5′-tetramethylbenzidine (0.1 mg/ml in 0.1M sodiumacetate, 0.1 M citric acid (pH 5.7] with 0.005% hydrogen peroxide) wasadded to each well and incubated for 30 min at 37° C. The reaction wasstopped by adding 1 ml of 4 M H₂SO₄ and the optical density was read ona Dynatech ELISA Reader (Dynatech Laboratories, Inc., Alexandria, Va.)at 450 nm. An optical density of >0.200 was considered positive.

[0096] SDS-PAGE and immunoblot analysis. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed bythe method of Tsang et al. (Tsang et al. (1983) Methods Enzymol.92:377-391), using an 8% acrylamide resolving gel. Equal volumes ofsample buffer (5% SDS-10% 2-mercaptoethanol-20% glycerol in 0.01 M TrisHCL, [pH 8.0]) and cell suspension containing 2.4 μg protein per μl weremixed, heated at 100° C. for 5 min, and a 5-μl portion was applied to 1of 15 wells. If the final protein content of the portion of sample to betested was <1.2 μg/μl, a volume up to 10 μl of sample was applied toachieve a final concentration of 6 μl of protein per well Proteinconcentrations were determined by the method of Markwell et al.(Markwell et al. (1978) Anal. Biochem. 87:206-210), with BSA as thestandard.

[0097] Proteins separated by SDS-PAGE were either silver stained by themethod of Morrissey (Morrissey, J. H. (1981) Anal. Biochem. 117:307-310)or electroblotted onto nitrocellulose (Schleicher & Schnell, Inc.,Keene, N. H.). The immunoblot procedure was done according to the methodof Tsang et al. (Tsang et al. (1983) Methods Enzymol. 92:377-391) withslight modifications. The blots were given three 5-min washes with PBS,pH 7.2, containing 0.3% Tween-20 and were gently agitated overnight (16h) at 25° C. The blots were blocked for 1 h with casein-thimerosalbuffer (CTB) (Kenna et al. (1985) J. Immunol. Meth. 85:409-419). Afterthree rinses with CTB, the blots were exposed to goat anti-mouseimmunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories,Richmond, Calif.) for 2 h at 25° C. Conjugate dilutions (1:2,000) weremade in CTB. The blots were again rinsed three times with CTB andexposed to 3-3′diaminobenzadine-4-hydrochloride in PBS, pH 7.2 (0.5mg/ml), with 0.003% H₂0₂ for 5 min at 25° C. Reactivity was expressed asa visible colored band on the nitrocellulose paper. Low molecular-massprotein standards (Bio-Rad) were used in PAGE and immunoblotting. Rabbitantisera to the protein standards were used to develop the standards(Carlone, G. M. (1986) Anal. Biochem. 155:89-91). Molecular masses werecalculated by the method of Neville and Glossman (Neville et al. (1974)Methods Enzymol. 32:92-102) using appropriate molecular mass standards.

[0098] IFA A bacterial suspension containing approximately 400-500 CFUper field (10 μl) was allowed to dry at room temperature on each well ofacetone-resistant, 12-well (5 mm diameter), glass slides (25×75 mm)(Cel-Line Associates, Newfield, N.J.). The slides were then immersed inacetone for 10 min and air dried at room temperature. MAbs were added tothe slides, which were incubated for 30 min at 37° C. After incubation,the slides were gently rinsed with PBS and soaked twice at 5-minintervals, blotted on filter paper, and air dried at room temperature.Fluorescein-labeled rabbit anti-mouse immunoglobulin (courtesy of W. F.Bibb, CDC) was then added, and the slides were incubated for 30 min at37° C. They were then washed twice with PBS and gently blotted on filterpaper. Slides were covered with carbonate-buffered mounting fluid, pH9.0, and cover slips and were then read with a Leitz Dialux 20fluorescence microscope equipped with a HBO-100 mercury incident lightsource, an I cube filter system, a 40× dry objective lens, and 6.3×binoculars (E. Leitz, Inc., Rockleigh, N.J.).

[0099] Immunoelectron-microscopy Pneumococcal cells were washed twotimes with PBS and fixed in a mixture of 1% paraformaldehyde-0.1%glutaraldehyde (freshly made) for 20 rnin at 4° C. The cells weredehydrated in a graded alcohol series and then in a 1:1 mixture ofabsolute ethanol and Lowicryl K4M (Ladd Research Industries, Inc.,Burlington, Vt.) for 1 h at 4° C. The cells were pelleted and suspendedin a 1:2 mixture of absolute ethanol and Lowicryl K4M for 1 h at 4° C.They were again pelleted and suspended in Lowicryl K4M (undiluted) for16 h at 4° C.

[0100] The cells were transferred to fresh Lowicryl K4M two times duringthe next 24-hour period. The Lowicryl K4M-treated cells were imbedded ingelatin capsules, which were placed inside a box lined with aluminumfoil. The capsules were hardened by holding them in the box 35 cm from ashort-wave UV light source for 72 h at −20° C. The box was brought toroom temperature, and the capsules were allowed to continue hardeningfor up to 14 days.

[0101] Samples of the capsule were cut into 100-μm thin sections andpicked up on nickel grids. Grids containing the sample were placed on adroplet of ovalbumin solution in PBS containing sodium azide (E. Y.Laboratories, Inc., San Mateo, Calif.) for 5 min. The grids (wet) weretransferred to a solution of primary MAbs diluted in a solution of BSAreagent (1% BSA in PBS containing 0.1% Triton X-100, Tween 20, andsodium azide) (E. Y. Laboratories) and incubated for 1 h at roomtemperature or 18 to 48 h at 4° C. in a moist chamber. For antibodybinding controls, other grids were wetted with MAbs against Legionellapneumophila. The grids were rinsed two times with PBS and incubated ondroplets of goat anti-mouse IgG-labeled colloidal gold particles (20μm)(E. Y. Laboratories) for 1 h at room temperature. The grids wererinsed two times and post-stained with osmium tetroxide, uranyl acetate,and lead citrate. The grids were examined with a Philips 410transmission electron microscope.

[0102] CBA/CaHN/J Mice X-linked immune deficiency (xid) of CBA/N mice asprepared by Wicker, L. S. and I. Seher, Curr. Top. Microbiol. Immunol.124:86-101 were used to study the protection afforded by the 37 kDaprotein.

Example 1

[0103] Monoclonal Antibodies

[0104] Hybridoma clone 1E7A3D7C2 produced MAbs that reacted with a37-kilodalton (kDa) protein antigen (pneumococcal fimbrial protein A)found in S. pneumoniae. The MAbs reacted with an antigen fractionated inSDS-PAGE, yielding a single immunoblot band. This indicates that the MAbreacted with epitopes found only on the 37-kDa antigen (pneumococcalfimbrial protein A). The MAbs produced by the immunization of mice withpneumococcal cells reacted with all pneumococcal strains tested (24serotypes) to yield a sensitivity of 100%. For specificity, 55 differentnonpneumococcal strains of bacteria that can also cause respiratoryinfections (Donowitz et al. (1985) In: Principles and practices ininfectious diseases, 2nd ed. (G. L. Mandell, R. G. Douglas, and J. E.Bennett, ed.) John Wiley & Sons, Inc., New York, pp. 394-404) weretested for antigens reacting with the MAbs. The latter strainsrepresented 19 genera and 36 species of bacteria. None of the strainstested reacted with the pneumococcal MAbs, thus yielding a specificityof 100%.

[0105] Of 44 patients known to have pneumococcus disease, 34 (77%) hadantibodies that reacted with the 37-kDa antigen (pneumococcal fimbrialprotein A) by Western immunoblot.

[0106] The MAbs reacted with whole pneumococcal cells to yield apositive test result in both the ELISA and IFA Results from both theELISA and the IFA indicate that the antigen has exposed epitopes on thesurface of the cell or that the immunoglobulin and other immunologicreagents are able to penetrate the pneumococcal cell walls.

[0107] Several strains of group A streptococci were tested forimmunofluorescence after reacting with the pneumococcus MAbs. None ofthe heterologous bacterial cells fluoresced in this test, indicatingthat the IFA reaction was specific for pneumococcus cells.

[0108] To further determine the location on the cell of the 37-kDaantigen (pneumococcal fimbrial protein A) epitopes reacting with theMAbs, immunolabeling experiments were performed. The cells were typicalof gram-positive cocci in the process of division A large portion of theantigen appears to be intracellular since there is no coating orlayering of the labeled MAbs around the cell. The large patch ofcolloidal gold staining indicates that the MAbs bound antigen locatedinside the cell wall. There was no colloidal gold binding to controlpneumococci that were exposed to the MAbs against L. pneumophila.

Example 2

[0109] Cloning of the Pneumococcal Fimbrial Protein A Gene Streptococcuspneumoniae DNA digested with restriction enzyme Sau3Al was ligated toBamHI digested pUC13 and transformed into E. coli TBI. Recombinantclones were identified by colony immunoblot using the 37-kDa monoclonalantibody. The plasmid pSTR3-1 is an example of the pneumococcal-fimbrialprotein A gene cloned into pUC13.

Example 3

[0110] Preparation of Purified 37 kDa Protein Antigen Two methods forpreparing the 37 kDa protein are used. (1) Streptococcus pneumoniae isconventionally cultured and the cells harvested. Purified 37 kDa proteinantigen (pneumococcal fimbrial protein A) is isolated from theStreptococcus pneumoniae cell mass by extraction with a non-ionicdetergent and further purified by ammonium sulfate fractionation andisoelectric focusing. (2) E. coli TBI strains containing plasmid pSTR3-1is cultured conventionally and the cells harvested. For improved yields,E. coli strains, transformed with an expression vector that carries astrong, regulated prokaryotic promoter and which contains the genecoding for the 37 kDa protein, is used. Suitable expression vectors arethose that contain a bacteriophage λPL Promoter (e.g., pKK1773-3), ahybrid trp-lac promoter (e.g., pET-3a) or a bacteriophage T7 promoter.The 37 kDa protein (PfpA) is then extracted from the separated cellmass.

[0111] Protection Experiments with 37 kDa Protein

[0112] Experiment No. 1

[0113] Twenty CBA/CaHN/J mice crying the xid (x-linked immunodeficiency)mutation were used in this protection study. They were tested forprotection against challenge with a virulent type 3 Streptococcuspneumoniae strain, WU2. Mice were anesthetized with Ketamine/Rompun andbled infraorbitally to obtain pre-immunization sera 37-kDa protein(pneumococcal fimbrial protein A) was emulsified in complete Freund'sadjuvant (CFA) to a protein concentration of 54 μg per ml. Ten mice wereinjected subcutaneously into 2 axillary and 2 inguinal sites at 0.1 mlper site, delivering approximately 22 μg protein/mouse. Ten control micewere treated identically with CFA and buffer substituting for protein.Fourteen days later, the ten test mice were injected intraperitoneally(IP) with 100 μg of the 37-kDa protein; controls were injected IP withbuffer. eight days following the IP immunizations, all mice were bledinfraorbitally to obtain post-immunization sera, and challengedintravenously (IV) with 60 CFU of a log phase culture of S. pneumoniaestrain WU2, a virulent capsular type 3 strain. Mice were observed for 21days, and deaths were recorded.

[0114] Sera were collected prior to immunizations to establish baselineexposures, and also following the full immunization protocol (but beforechallenge) in order to correlate circulating antibody to the 37 kDaprotein with protection.

[0115] Days post challenge:

[0116] 1—no deaths

[0117] 2—3 control mice dead

[0118] 3—2 control mice dead

[0119] 4—2 control mice dead, one sick

[0120] 5—1 control mouse dead

[0121] 6-21 no deaths

[0122] Immunized with 37 kDa protein: 10/10 survived

[0123] Controls with no protein: 2/19 survived (8/10 died)

[0124] Difference statistically significant: (p=0.0008) Rank sum test

[0125] Experiment No. 2

[0126] Twenty CBA/CaHN/J mice carrying the xid mutation were injectedaccording to the following protocol:

[0127] 1. All mice were bled prior to immunization to establish baselineimmunity. Ten test mice were immunized subcutaneously in four sites witha total of 21 pg of 37-kDa protein antigen (pneumococcal fimbrialprotein A) emulsified in Complete Freund's adjuvant (CFA). Ten controlmice were immunized identically with CFA and buffer substituting for theantigen.

[0128] 2. Fourteen days later, the mice were boosted intraperitoneally(I.P.) with 100 pg of the 37 kDa protein antigen (test mice) or withbuffer (controls). No adjuvant was used with this booster immunization.

[0129] 3. Eight days later, all mice were bled via the infraorbitalsinus and the sera were collected and pooled into the two groups(immunized and controls). At the same time, blood was collected fromindividual mice to assay for antibody responses.

[0130] 4. One day later, two additional mice were injected I.O. with 0.1ml of pooled immune sera to attempt to passively transfer immunity.Three additional mice were injected I.P. with 0.1 ml of pooled controlmouse sera. (only five mice were injected at this step because of thesmall amount of sera obtained from the immunized mice.).

[0131] 5. One hour after the I.P. injections, these five mice werechallenged intravenously (I.V.) with 140 colony-forming units (CFU) of amid-log phase pneumococcal type 3 strain, WU2.

[0132] 6. At the same time, the eighteen (8 test and 10 control)* micewere challenged I.V. with the same culture of WU2.

[0133] 7. Deaths were tallied daily. RESULTS: No. Dead/No. ChallengedImmunized with the 37 kDa protein:  0/8* Control mice: 10/10 PassiveProtection: mice receiving immune sera: 0/2 Mice receiving control sera:3/3

[0134] Mice immunized with the 37 kDa protein were protected from fatalchallenge with strain WU2, and this immunity could be passivelytransferred with sera from immunized mice.

[0135] Experiment No. 3

[0136] An enzyme-linked immunosorb6nt assay (ELISA) was developed usingpurified S. pneumoniae 37-kDa protein antigen as a capture for humanantibodies. Paired sera were tested from children, less than 24 monthsof age, known to have pneumococcal pneumonia. Disease confirmation wasdetermined by blood culture or antigen in the urine. It was found that35% (9/26) had antibody titers greater than serafrom non-ill children ofthe same age group, p=0.06. This illustrates that some of the childrenresponded to the 37-kDa protein antigen after natural infection.

[0137] Preparation of the 37 kDa Protein or Polypeptide Conjugate

[0138] Conjugates can be prepared by use of a carrier protein bound tothe 37-kDa protein or polypeptides derived from the 37-kDa protein via alinker, to elicit a T cell dependent response. Such carrier proteinscould be any immunogenic protein, for example, keyhole limpethemocyanin, bovine serum albumin, tetanous toxoid, diphtheria toxoid,and bacterial outer membrane proteins. Examples of bacterial outermembrane proteins, useful as conjugates; include outer membrane proteinsof Neisseria meningitides and Haemophilus influenzae. Neisseriameningitides can be an organism selected from Neisseria meningitides,group A, B, or C.

[0139] In addition, the 37-kDa protein or polypeptides thereof can beused in a conjugate where the 37-kDa protein or polypeptides thereof arethe T-cell dependent immunogenic carrier for polysaccharide antigensthat are B-cell stimulators. This is based on the theory thatpolysaccharide antigens are B-cell stimulators and that protectiveimmunity is usually generated by a combination of B-cell and T-cellstimulation. Protein antigens exhibit T-cell dependent properties; i.e.,booster and carrier priming. T-cell dependent stimulation is importantbecause children less than two years of age do not respond to T-cellindependent antigens. The attachment or conjugation of antigens can beaccomplished by conventional processes, such as those described in U.S.Pat. No. 4,808,700, involving the addition of chemicals that enable theformation of covalent chemical bonds between the carrier immunogen andthe immunogen.

[0140] In use, the 37-kDa protein antigen of this invention can beadministered to mammals; e.g., human, in a variety of ways. Exemplarymethods include parenteral (subcutaneous) administration given with anontoxic adjuvant, such as an alum precipitate or peroral administrationgiven after reduction or ablation of gastric activity; or in apharmaceutical form that protects the antigen against inactivation bygastric juice (e.g., a protective capsule or microsphere).

[0141] The dose and dosage regimen will depend mainly upon whether theantigen is being administered for therapeutic or prophylactic purposes,the patient, and the patient's history. The total pharmaceuticallyeffective amount of antigen administered per dose will typically be inthe range of about 2 μg to 50 μg per patient.

[0142] For parenteral administration, the antigen will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutically acceptable parenteralvehicle. Such vehicles are inherently nontoxic and nontherapeutic.Examples of such vehicles include water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Non aqueous vehicles,such as fixed oils and ethyl oleate, may also be used. Liposomes may beused as vehicles. The vehicle may contain minor amounts of additives,such as substances which enhance isotonicity and chemical stability;e.g., buffers and preservatives.

Example 4

[0143] Bacterial strains. All isolates of S. pneumoniae were providedand serotyped by the Streptococcal Reference Laboratory, Division ofBacterial and Mycotic Diseases, NCID, Centers for Disease Control andPrevention (CDC). The pneumococcal serotype 6B strain used for cloningand sequencing was a CDC reference strain (SP-86). E. Coli DH5α(Bethesda Research Laboratories, Gaithersburg, Md.) was used as therecipient host for plasmids, pUC 19 and its derivatives.

[0144]S. pneumoniae strains were grown on Trypticase soy agar plateswith 5% sheep blood cells or, where indicated, in Todd-Hewitt brothcontaining 0.5% yeast extract. E. coli cultures were grown in Luriabroth which, when required, was supplemented with 100 μg/nil ofampicillin (Sigma Chemical Co., St. Louis, Mo.).

[0145] Cloning and Sequencing of the psaA Gene from S. pneumoniae,Serotype 6b.

[0146] A chromosomal library from S. pneumoniae serotype 6B was preparedas previously described (Sampson et al. 1994. Cloning and nucleotidesequence analysis of psaA, the Streptococcus pneumoniae gene encoding a37-kilodalton protein homologous to previously reported Streptococcussp. adhesins. Infect. Immun 62:319-324.), except that pUC18 was used asthe cloning vector instead of pUC 13. Recombinants were screened bycolony immunoblot using monoclonal antibody (MAb) 1E7. (Russell et al.1990. Monoclonal antibody recognizing a species-specific protein fromStreptococcus pneumoniae. J. Clin. Microbiol. 28:2191-2195). Thisprocedure, as well as plasmid purification from positive clones(Ish-Horowicz et al. 1981. Rapid and efficient cosmid cloning. NucleicAcids Res. 9:2989-2998.) and restriction endonuclease analysis, has allbeen previously described. (Sampson et al. 1990. Nucleotide sequence ofhtpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa)common antigen, formerly designated the 60-kDa common antigen. Infect.Immun. 58:3154-3157 and Sampson et al. 1994). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotanalysis were done as before (Sampson et al. 1990). All other DNAmanipulations were done according to methods described in Sambrook etal. DNA sequencing was performed using the ABI PRISM Dye TerminatorCycle Sequencing kit and procedure (Perkin-Elmer, Cetus, Foster City,Calif.). Sequence data were analyzed with the DNASTAR software program(DNASTAR Inc., Madison, Wis.) and the Wisconsin Genetics Computer Groupsequence analysis software program (Fenno et al. 1989. Nucleotidesequence analysis of a type 1 fimbrial gene of Streptococcus sanguisFW213. Infect Immun. 57:3527-3533).

[0147] Preparation of genomic DNA for PCR-RFLP analysis. High molecularweight pneumococcal DNA was prepared by the procedure of Graves andSwaminathan (Graves et al. 1993. Universal bacterial DNA isolationprocedure, p. 617-621. In D. H. Pershing et al. (ed), Diagnosticmolecular biology. American Society for Microbology, Washington, D.C.)with modifications. Sixteen-hour cultures of type specific S. pneumoniaewere grown in 50 ml of Todd-Hewitt broth containing 0.5% yeast extractin screw cap flasks at 37° C. without shaking. Cultures were pelleted at8000×g for 15 min at room temperature and washed with phosphate-bufferedsaline (10 mM, pH 7.2). The cell pellet was solubilized in 2.5 ml ofbuffer composed of 10 mM Tris, 1.0 mM EDTA, pH 8.0, and 0.4% SDS.Fifteen microliters of proteinase K (20 mg/ml) was added, and the lysatewas incubated at 37° C. for 1 h. The mixture was adjusted to 0.48 M NaClwith the addition of 500 μl of 5M NaCl and, after mixing by inversion,400 μl of 10% hexadecyltriethylaunonium bromide in 0.7% NaCl was added.This suspension was mixed as before, incubated for 30 min at 65° C., andextracted with an equal volume of phenol-chloroform-isoamyl alcohol. Theupper aqueous phase was separated by centrifugation at 1500×g andextracted with chloroform-isoamyl alcohol. DNA was precipitated from theupper aqueous phase with 2.5 volumes of ethanol at −70° C. for 30 min.It was pelleted and dried in a desiccator, resuspended in water andquantitated by measuring absorbance at 260 nm.

[0148] PCR-RFLP. Restriction enzymes EcoRI, HinfI, MaeIII, MboII, MnlI,and NheI were obtained from Boerhringer Mannheim Biochemicals(Indianapolis, Id.); RsaI, Tsp509I, Eco57I, and XmnI were purchased fromNew England Biolabs (Beverly, Mass.). Primer sequences for theamplification reaction were selected from the N-terminal (nucleotides181-201) and C-terminal (nucleotides 1106-1126) sequences of the S.pneumoniae serotype 6B gene (P1, AGGATCTAATGAAAAAATTAG (SEQ ID NO:3);P2, TCAGAGGCTTATTTTGCCAAT (SEQ ID NO:4)) and flanking regions. Theprimers were synthesized at the Centers for Disease Control andPrevention using standard procedures.

[0149] (i) DNA amplification. The reaction was performed with thePerkin-Elmer PCR amplification kit. Reaction volumes were 100 μl andcontained the standard 1× reaction buffer without Mg, 1 μM of eachprimer, 2.0 mM MgCl₂, 0.2 mM dNTPs, template DNA, and 2.5 U of Taq DNApolymerase. The source of the template DNA was either extracted purifiedchromosomal DNA or a bacterial colony. Conditions for amplification wereas follows: 30 cycles of denaturation 94° C., 1 min., annealing 52° C.,0.5 min., and extension 72° C., 1.5 min. Amplified products wereseparated on a 1% agarose gel and visualized with ethidium bromide. Adirect colony amplification procedure was adapted, which shortenedtemplate preparation by eliminating the necessity of extractingchromosomal DNA. The procedure consisted of adding a single bacterialcolony directly from the plate into the PCR reaction mixture and heatingat 95 ° C. for 10 minutes. The remaining PCR steps were performed asoutlined for extracted chromosomal DNA and are given above.

[0150] (ii) Enzyme digestion. Digestion of amplified products wasperformed as directed by the manufacturer for the designated enzymes involumes of 20 μl. Digestion products were analyzed by agarose (2%Metaphor agarose, FMC Corp., Rockland, Me.) gel electrophoresis andvisualized after being stained with ethidium bromide

[0151] Analysis of Type 6B psaA.

[0152] Genomic DNA was partially digested by Sau3AI was ligated toBamHI-digested pUC18 and used to transform E. coli DH5α. Recombinantcolonies were selected for resistance to ampicillin and the formation ofwhite colonies in the presence of isopropyl-β-D-galactopyranoside (IPTG)and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Colony immunoblotscreening (using anti-PsaA MAb) of approximately 2,500 colonies yieldedtwo positive clones, which were selected, purified, and rescreened byWestern blot analysis using the same MAb. They both expressed a proteinreactive with MAb to PsaA and which migrated in SDS-PAGE with theexpected molecular mass of approximately 37 kDa. One was selected forcontinued study and was designated pSTR6.

[0153] Limited restriction enzyme analysis of DNA from the recombinantplasmid showed that the positive clone contained an insert that was 3.5kb with sites for enzymes ClaI, EcoRI, and HindIII. To localize the PsaAcoding region, the insert was double digested with SstI (multiplecloning site in vector) and HindIII. The resultant fragments wereligated into pUC18 and transformed into E. coli DH5α. This generated arecombinant containing an insert of 1.3 kb in size. The resultantsubclone pSTR6y, when analyzed by SDS-PAGE and Western blot usinganti-PsaA MAb, was shown to express full length PsaA immuno-reactiveprotein.

[0154] The complete nucleotide sequence on both strands of the 1.3-kbinsert was determined by cycle sequencing of the plasmid subclone usingoligonucleotide primers complementary to the sequence. These were madeas sequence information became available. The nucleotide sequence of theentire streptococcal insert is set forth in the Sequence Listing as SEQID NO:1. The single open reading frame (ORF) present, beginning atnucleotide (nt) 189 and ending at nt 1117, encodes the psaA genesequence. This ORF is 930 nt long and when amplified and subcloned intovector systems such as pGEM (Promega, Madison, Wis.) and BAC-to-BAC™expression system (Bethesda Research Laboratories, Gaithersburg, Md.)expresses full-length PsaA, reactive with anti-PsaA MAb antibodies. ThisORF encodes a peptide of 309 amino acids with a deduced molecular weightof 34,598 and an isoelectric point of 5.23. Analysis of the peptideusing the algorithm of Kyte and Doolittle (Kyte et al. 1982. “A simplemethod for displaying the hydropathic character of a protein.” J. Mol.Biol. 157:105-132) shows that the peptide contains a major hydrophobicregion of 20 amino acids which encodes a putative leader sequence. Thisleader contains the consensus sequence for signal peptidase cleavage(LXXC). Removal of this leader would result in a peptide of molecularmass 32,465 with a predicted isoelectric point of 4.97. A consensussequence for a ribosomal binding site (Shine et al. 1974. “The3′-terminal sequence of E. coli 16S ribosomal RNA: complementarity tononsense triplets and ribosomal binding sites.” Proc. Natl. Acad. Sci.USA 71:1324-1346) is located 5 nt upstream of the ATG start codon.

[0155] Comparison of the Serotype 6B Sequence with StreptococcalHomologs.

[0156] Comparison of the serotype 6B psaA nucleotide sequence ((Bilofskyet al. 1988. A GenBank genetic sequence database. Nucleic Acids Res.16:1861-1864) GenBank accession number U53509) and its flanking regionswith the previously published strain R36A psaA sequence (Sampson et al.1994. “Cloning and nucleotide sequence analysis of psaA, theStreptococcus pneumoniae gene encoding a 37-kilodalton proteinhomologous to previously reported Streptococcus sp. adhesins.” Infect.Immun. 62:319-324) shows the differences between the nucleotidesequences. The computed homology between the two sequences is 74%. Majorareas of discord are in regions upstream and downstream of the ORF andin the initial 60 nt which encode the putative signal peptide. When thetwo PsaA coding sequences are compared the sequence homology increasesto 78%. Serotype 6B sequence was also compared to thepsaA DNA sequencefor another vaccine serotype, serotype 2, which was recently submittedto GenBank by Berry and Paton (Accession number U40786). Computeranalysis of these two sequences shows that they are very similar, withcomputed DNA homology percentages of 99% between the two psaA DNAsequences. There are eight single base differences between the twosequences.

[0157] A comparison of serotype 2 and 6B PsaAs shows almost completeidentity: the computed similarity value is 99.3. The eight basedifference at the nucleotide level translated into a difference at thepeptide level of six amino acids with two of the changes resulting inconservative substitutions. Further analyses and comparisons of theserotype 6B sequence to the other five GenBank PsaA homologues fromviridans Streptococci and E. faecalis (Fenno et al. 1989. “Nucleotidesequence analysis of a type 1 fimbrial gene of Streptococcus sanguisFW213.” Infect. Immun. 57:3527-3533, Sampson et al. 1994. “Cloning andnucleotide sequence analysis of psaA, the Streptococcus pneumoniae geneencoding a 37-kilodalton protein homologous to previously reportedStreptococcus sp. adhesins.” Infect. Immun. 62:319-324, Ganeshkumar etal. 1991. “Nucleotide sequence of a gene coding for a salvia-bindingprotein (SsaB) from Streptococcus sanguis 12 and possible role of theprotein in coaggregation with actinomyces.” Infect. Immun 59:1093-1099,Kolenbrander et al. 1994. “Nucleotide sequence of the Streptococcusgordonii PK488 coaggregation adhesin gene scaA and ATP-bindingcassette.” Infect. Immun. 62:4469-4480, and Lowe et al. 1995. “Cloningof an Enterococcus faecalis endocarditis antigen: homology with someadhesins from oral streptococci.” Infect. Immun 63:703-706) revealedsignificant sequence similarity between them. Sequence identities were81%, 81%, 77%, 82%, and 57%, respectively, for PsaA (S. pneumoniaestrain R36A), SsaB (S. sanguis), FimA (S. parasanaguis), ScaA (S.gordonii) and EfaA (E. faecalis). Additionally, all six sequences showedgreat siarity in organization. They have a hydrophobic leader peptidecontaining the prolipoprotein consensus sequence LXXC (for signalpeptidase II cleavage) within the first 17-20 amino acids. ThisN-terminal leader sequence appears to represent the area of greatestvariability. It is followed by a region of high similarity from aminoacids 36-150. The region from 150 to 198 is a variable region and isfollowed by another conserved region (198-309).

[0158] PCR-RFLP Analysis of Chromosomal DNA from the 23 Serotype Strainsin a 23-Valent Vaccine.

[0159] PCR-RFLP was used to examine the degree of conservation of thegene among 23 S. pneumoniae serotypes, representing the 23 serotypes ina 23-valent vaccine. Since previous attempts to amplify pneumococcaltype strains with primers corresponding to strain R36A wereunsuccessful, primers for PCR were selected from N-terminal andC-terminal sequences of serotype 6B. Using primers complementary toserotype 6B, the psaA gene from all 23 serotypes and subtypesrepresented in the 23-valent vaccine was amplified from chromosomal DNA.A total of 10 enzymes were chosen that had restriction endonucleasedigestion sites throughout the entire length of the serotype 6B psaAgene. Nine of the 10 enzymes gave identical patterns for all 23 psaAgenes analyzed.

[0160] Cleavage with restriction enzyme Tsp509I was the one exception tothose enzymes that generated identical patterns. Tsp509I has six siteswithin the gene and generates seven fragments upon digestion with sizesof 7, 30, 68, 146, 151, 166, and 362 bp. When these fragments areseparated on 2% metaphor agarose gel, a five-band pattern can be seen(7- and 30-bp fragments are not seen on these gels because of theirsmall size). For 21 of 23 serotypes this five-fragment enzyme patternwas obtained; but for strains of serotype 4 and 33F, the 146-bp fragmentis absent and two new fragments appear flanking the 68-bp fragmentmaking a total of seven bands. This increase in fragment number resultsfrom the presence of an extra Tsp509I site within the 146-bp fragment.

[0161] To ascertain the prevalence of this extra site, the Tsp509Ipatterns of 3 to 4 additional strains of each of 23 serotype strains(additional strains of serotype 2 and serotype 25 were not available)were analyzed. All strains analyzed were random clinical isolates fromthe United States that had been submitted to CDC for serotyping. Themajority of the 80 strains were blood isolates; exceptions were 2 fromcerebrospinal fluid, 2 from pleural fluid, and 1 each from the eye andnose. Of the strains analyzed, 10% had the extra Tsp509I site, resultingin the altered RFLP pattern. This modification was seen only in types 4,8, 11F, and 33F. In an attempt to determine prevalence of this alteredpattern, we analyzed the psaA gene from 8 additional strains of these 4types for the Tsp509I variation (bringing the total to 11-12 for these 4types). Table 1 summarizes the analyses of serotypes 4, 8, 11A, and 33F;it shows that the modified pattern is randomly present in 4 and 8, butis present in 11 of 12 strains of 11A and all strains of 33F. Theoccurrence of this pattern could not be correlated with geographiclocation or region of the United States since strains that showedvariation came from diverse regions of the country. All strains of types4, 8, 11A, and 33F were blood isolates except one 33F strain, which wasa nasal isolate; thus the relevance of the site of isolation onprevalence of this modification could not be assessed. TABLE 1 Screeningof selected serotypes for additional Tsp509I restriction site Ratio ofserotypes with additional site to Total serotypes with total no. ofserotypes tested unique patterns Serotype Expt. #1^(a) Expt. #2^(b) %Unique pattern 4 1/3 3/9 33 (4/12)^(c) 8 3/4 4/9 44 (7/13) 11A 2/3 9/992 (11/12) 33F 3/3 9/9 100 (12/12)

[0162] This analysis discloses the cloning and sequencing of the geneencoding PsaA from S. pneumoniae serotype 6B and a subsequent analysisof the gene in the 23 pneumococcal polysaccharide vaccine serotypes.Sequence analysis revealed that the serotype 6B sequence and thepreviously published strain R36A were less similar than expected. Thenucleotide sequence and its flanking regions were only 73% homologous tothe original strain R36A psaA, with the actual PsaA coding sequences hada computed homology of 78%. Protein sequence similarity between the twosequences was only 81%. A comparison of the serotype 6B sequence withthe newly submitted serotype 2 pneumococcal psaA (a vaccine serotype)gave computed DNA homology values of 99% and 98% protein sequencesimilarity. These values are evidence of the high sequence conservationfor the gene within the vaccine serotypes. Moreover, when the deducedamino acid sequences of these two sequences were compared with otherpublished sequences for PsaA homologues within the genus, large areas ofsimilarity were evident for all five proteins. Similarity values withinthe group ranged from 57% to 82%.

[0163] The need for a Streptococcus pneumoniae vaccine candidateprompted us to clone and sequence the psaA gene from S. pneumoniaeserotype 6B. The heterogeneity between the two pneumococcal psaA genes(6B and R36A) led us to examine the vaccine serotypes to determine thedegree of diversity among strains. Primers homologous with the Nterminus and C terminus of the serotype 6B sequence amplified all 23 ofthe vaccine serotypes. PCR-RFLP analysis using 10 different restrictionenzymes representing 21 sites within the serotype 6B gene and shows onlyone area of diversity, which resulted in an additional Tsp509I site fora small number of strains.

[0164] This study demonstrates that the serotype 6B gene sequence isrepresentative of the sequence found among the vaccine serotypes.Evidence for this includes the 99% DNA sequence identity betweenserotype 2 and serotype 6B and the uniform and identical restrictionpatterns covering the 21 sites examined in this study. It is clear thatour earlier strain R36A psaA sequence represents a variant sequenceseemingly not present in the serotypes that were analyzed here since wewere unable to amplify them using primers to strain R36A psaA.

[0165] The more important aspect of this study, however, is that thereis limited diversity among the vaccine serotypes analyzed. These are theserotypes that cause disease and thus, the ones against whichprophylactic measures are needed. The lack of genetic diversity of psaAamong these serotypes suggests that gene is highly conserved and is anexcellent candidate for vaccine development.

[0166] The 37-kDa protein from serotype 22F was used to generatemonoclonal antibodies 1B6E12H9, 3C4D5C7, 4E9G9D3, 4H5C10F3, 6F6F9C8,8G12G11B10, which were analyzed for their ability to confer protectionfrom infection by Streptococcus pneumoniae. Table 2 shows that of 5monoclonal antibodies tested, one in particular gave efficientprotection from subsequent S. pneumoniae challenge (8G12G11B10). Theprotection from S. pneumoniae was dose-responsive, demonstrating thatthe monoclonal antibody was responsible for the protection (Table 3).TABLE 2 Passive protection of five (5) Anti-37 kDa murine monoclonalantibodies in an infant mouse model to Streptococcus pneumoniae serotype6B. Death 37 kDa MAb Bacteremia @ 48 h @ 14 d Cell Line^(a) @ 48 h (%)(%) (%) 1E7 . . . 100 100 100 8G12 . . . 100 0 20 4E9 . . . 100 80 1006F6 . . . 100 60 100 1B6 . . . 100 80 100

[0167] TABLE 3 Effect of a Second Dose on the Passive ProtectivePotential of the Anti- 37 kDa Murine Monoclonal Antibody 8G12. Ab DoseLevel Bacteremia Death (μg) @ 48 h @ 48 h @ 10 d Pre Post^(a) % Avecfu/ml (%) (%) 50 — 100 1.2 × 10⁴ 0 30 50 50 80 1.0 × 10⁴ 0 50  5 — 1004.7 × 10⁴ 70 100  5  5 100 3.0 × 10⁴ 50 80 — — 100 >10⁵ 80 100

[0168]

1 4 1 1330 DNA STREPTOCOCCUS PNEUMONIAE CDS (189)...(1115) 1 tactgcttcagttttgggac tctttattgg ctatagtttt aatgttgcgg caggttctag 60 tatcgtgcttacagctgcta gtttctttct cattagcttc tttatcgctc ccaaacaacg 120 atatttgaaactgaaaaata aacatttgtt aaaataaggg gcaaagccct aataaattgg 180 aggatcta atgaaa aaa tta ggt aca tta ctc gtt ctc ttt ctt tct gca 230 Met Lys Lys LeuGly Thr Leu Leu Val Leu Phe Leu Ser Ala 1 5 10 atc att ctt gta gca tgtgct agc gga aaa aaa gat aca act tct ggt 278 Ile Ile Leu Val Ala Cys AlaSer Gly Lys Lys Asp Thr Thr Ser Gly 15 20 25 30 caa aaa cta aaa gtt gttgct aca aac tca atc atc gct gat att act 326 Gln Lys Leu Lys Val Val AlaThr Asn Ser Ile Ile Ala Asp Ile Thr 35 40 45 aaa aat att gct ggt gac aaaatt gac ctt cat agt atc gtt ccg att 374 Lys Asn Ile Ala Gly Asp Lys IleAsp Leu His Ser Ile Val Pro Ile 50 55 60 ggg caa gac cca cac gaa tac gaacca ctt cct gaa gac gtt aag aaa 422 Gly Gln Asp Pro His Glu Tyr Glu ProLeu Pro Glu Asp Val Lys Lys 65 70 75 act tct gag gct gat ttg att ttc tataac ggt atc aac ctt gaa aca 470 Thr Ser Glu Ala Asp Leu Ile Phe Tyr AsnGly Ile Asn Leu Glu Thr 80 85 90 ggt ggc aat gct tgg ttt aca aaa ttg gtagaa aat gcc aag aaa act 518 Gly Gly Asn Ala Trp Phe Thr Lys Leu Val GluAsn Ala Lys Lys Thr 95 100 105 110 gaa aac aaa gac tac ttc gca gtc agcgac ggc gtt gat gtt atc tac 566 Glu Asn Lys Asp Tyr Phe Ala Val Ser AspGly Val Asp Val Ile Tyr 115 120 125 ctt gaa ggt caa aat gaa aaa gga aaagaa gac cca cac gct tgg ctt 614 Leu Glu Gly Gln Asn Glu Lys Gly Lys GluAsp Pro His Ala Trp Leu 130 135 140 aac ctt gaa aac ggt att att ttt gctaaa aat atc gcc aaa caa ttg 662 Asn Leu Glu Asn Gly Ile Ile Phe Ala LysAsn Ile Ala Lys Gln Leu 145 150 155 agc gcc aaa gac cct aac aat aaa gaattc tat gaa aaa aat ctc aaa 710 Ser Ala Lys Asp Pro Asn Asn Lys Glu PheTyr Glu Lys Asn Leu Lys 160 165 170 gaa tat act gat aag tta gac aaa cttgat aaa gaa agt aag gat aaa 758 Glu Tyr Thr Asp Lys Leu Asp Lys Leu AspLys Glu Ser Lys Asp Lys 175 180 185 190 ttt aat aag atc cct gct gaa aagaaa ctc att gta acc agc gaa gga 806 Phe Asn Lys Ile Pro Ala Glu Lys LysLeu Ile Val Thr Ser Glu Gly 195 200 205 gca ttc aaa tac ttc tct aaa gcctat ggt gtc cca agt gcc tac atc 854 Ala Phe Lys Tyr Phe Ser Lys Ala TyrGly Val Pro Ser Ala Tyr Ile 210 215 220 tgg gaa atc aat act gaa gaa gaagga act cct gaa caa atc aag acc 902 Trp Glu Ile Asn Thr Glu Glu Glu GlyThr Pro Glu Gln Ile Lys Thr 225 230 235 ttg gtt gaa aaa ctt cgc caa acaaaa gtt cca tca ctc ttt gta gaa 950 Leu Val Glu Lys Leu Arg Gln Thr LysVal Pro Ser Leu Phe Val Glu 240 245 250 tca agt gtg gat gac cgt cca atgaaa act gtt tct caa gac aca aac 998 Ser Ser Val Asp Asp Arg Pro Met LysThr Val Ser Gln Asp Thr Asn 255 260 265 270 atc cca atc tac gca caa atcttt act gac tct atc gca gaa caa ggt 1046 Ile Pro Ile Tyr Ala Gln Ile PheThr Asp Ser Ile Ala Glu Gln Gly 275 280 285 aaa gaa ggc gac agc tac tacagc atg atg aaa tac aac ctt gac aag 1094 Lys Glu Gly Asp Ser Tyr Tyr SerMet Met Lys Tyr Asn Leu Asp Lys 290 295 300 att gct gaa gga ttg gca aaataagcctctg aaaaacgtca ttctcatgtg 1145 Ile Ala Glu Gly Leu Ala Lys 305agctggcgtt ttttctatgc ccacatttcc ggtcaaatca ttggaaaatt ctgactgttt 1205cagatacaat ggaagaaaaa agattggagt atcctatggt aacttttctc ggaaatcctg 1265tgagctttac aggtaaacaa ctacaagtcg gcgacaaggc gcttgatttt tctcttacta 1325caaca 1330 2 309 PRT STREPTOCOCCUS PNEUMONIAE 2 Met Lys Lys Leu Gly ThrLeu Leu Val Leu Phe Leu Ser Ala Ile Ile 1 5 10 15 Leu Val Ala Cys AlaSer Gly Lys Lys Asp Thr Thr Ser Gly Gln Lys 20 25 30 Leu Lys Val Val AlaThr Asn Ser Ile Ile Ala Asp Ile Thr Lys Asn 35 40 45 Ile Ala Gly Asp LysIle Asp Leu His Ser Ile Val Pro Ile Gly Gln 50 55 60 Asp Pro His Glu TyrGlu Pro Leu Pro Glu Asp Val Lys Lys Thr Ser 65 70 75 80 Glu Ala Asp LeuIle Phe Tyr Asn Gly Ile Asn Leu Glu Thr Gly Gly 85 90 95 Asn Ala Trp PheThr Lys Leu Val Glu Asn Ala Lys Lys Thr Glu Asn 100 105 110 Lys Asp TyrPhe Ala Val Ser Asp Gly Val Asp Val Ile Tyr Leu Glu 115 120 125 Gly GlnAsn Glu Lys Gly Lys Glu Asp Pro His Ala Trp Leu Asn Leu 130 135 140 GluAsn Gly Ile Ile Phe Ala Lys Asn Ile Ala Lys Gln Leu Ser Ala 145 150 155160 Lys Asp Pro Asn Asn Lys Glu Phe Tyr Glu Lys Asn Leu Lys Glu Tyr 165170 175 Thr Asp Lys Leu Asp Lys Leu Asp Lys Glu Ser Lys Asp Lys Phe Asn180 185 190 Lys Ile Pro Ala Glu Lys Lys Leu Ile Val Thr Ser Glu Gly AlaPhe 195 200 205 Lys Tyr Phe Ser Lys Ala Tyr Gly Val Pro Ser Ala Tyr IleTrp Glu 210 215 220 Ile Asn Thr Glu Glu Glu Gly Thr Pro Glu Gln Ile LysThr Leu Val 225 230 235 240 Glu Lys Leu Arg Gln Thr Lys Val Pro Ser LeuPhe Val Glu Ser Ser 245 250 255 Val Asp Asp Arg Pro Met Lys Thr Val SerGln Asp Thr Asn Ile Pro 260 265 270 Ile Tyr Ala Gln Ile Phe Thr Asp SerIle Ala Glu Gln Gly Lys Glu 275 280 285 Gly Asp Ser Tyr Tyr Ser Met MetLys Tyr Asn Leu Asp Lys Ile Ala 290 295 300 Glu Gly Leu Ala Lys 305 3 21DNA UNKNOWN PRIMER 3 aggatctaat gaaaaaatta g 21 4 21 DNA UNKNOWN PRIMER4 tcagaggctt attttgccaa t 21

What is claimed is:
 1. An isolated nucleic acid encoding the 37-kDaprotein of Streptococcus pneumoniae as set forth in the Sequence Listingas SEQ ID NO:2.
 2. The isolated nucleic acid of claim 1, wherein thenucleic acid is the nucleic acid set forth in the Sequence Listing asSEQ ID NO:1.
 3. An isolated nucleic acid comprising a unique fragment ofat least 10 nucleotides of the nucleic acid set forth in the SequenceListing as SEQ ID NO:
 1. 4. An isolated nucleic acid comprising thenucleic acid as set forth in the Sequence Listing as SEQ ID NO:3.
 5. Anisolated nucleic acid comprising the nucleic acid as set forth in theSequence Listing as SEQ ID NO:4.
 6. The isolated nucleic acid of claim 1in a vector suitable for expressing the nucleic acid.
 7. The vector ofclaim 6 in a host suitable for expressing the nucleic acid.
 8. Theisolated nucleic acid of claim 3 in a vector suitable for expressing thenucleic acid.
 9. The vector of claim 8 in a host suitable for expressingthe nucleic acid.
 10. A purified polypeptide encoded by the nucleic acidof claim
 2. 11. A purified polypeptide encoded by the nucleic acid ofclaim
 3. 12. A purified antibody which selectively binds with thepolypeptide of claim
 10. 13. A purified antibody which selectively bindswith the polypeptide of claim
 11. 14. A vaccine comprising animmunogenic polypeptide of claim 10 and a pharmaceutically acceptablecarrier.
 15. A vaccine comprising an immunogenic polypeptide of claim 11and a pharmaceutically acceptable carrier.
 16. A method of detecting thepresence of the Streptococcus pneumoniae in a sample, comprising thesteps of: a) contacting a sample suspected of containing Streptococcuspneumoniae with nucleic acid primers capable of hybridizing to a nucleicacid comprising a unique portion of the nucleic acid of claim 2; b)amplifying the nucleic acid comprising a portion of the nucleic acid ofclaim 2; c) detecting the presence of an amplification product, thepresence of the amplification product indicating the presence ofStreptococcus pneumoniae in the sample.
 17. A method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof: a) contacting an antibody-containing sample from the subject withthe polypeptide of claim 10; and b) detecting the binding of theantibody with the polypeptide, the binding indicating the presence ofStreptococcus pneumoniae in the subject.
 18. A method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof: a) contacting an antibody-containing sample from the subject withthe polypeptide of claim 11; and b) detecting the binding of theantibody with the polypeptide, the binding indicating the presence ofStreptococcus pneumoniae in the subject.
 19. A method of detecting thepresence of Streptococcus pneumoniae in a subject, comprising the stepsof: a) contacting a sample from the subject with the antibody of claim12; and b) detecting the binding of the antibody with an antigen, thebinding indicating the presence of Streptococcus pneumoniae in thesubject.
 20. A method of detecting the presence of Streptococcuspneumoniae in a subject, comprising the steps of: a) contacting a samplefrom the subject with the antibody of claim 13; and b) detecting thebinding of the antibody with an antigen, the binding indicating thepresence of Streptococcus pneumoniae in the subject.
 21. A method ofpreventing Streptococcus pneumoniae infection in a subject, comprisingadministering to the subject a prophylactically effective amount of thevaccine of claim 14 and a pharmaceutically acceptable carrier.
 22. Amethod of preventing Streptococcus pneumoniae infection in a subject,comprising administering to the subject a prophylactically effectiveamount of the vaccine of claim 15 and a pharmaceutically acceptablecarrier.
 23. A method of preventing Streptococcus pneumoniae infectionin a subject, comprising administering to the subject a prophylacticallyeffective amount of an anti-idiotype antibody to the polypeptide ofclaim 10 and a pharmaceutically acceptable carrier.
 24. A method ofpreventing Streptococcus pneumoniae infection in a subject, comprisingadministering to the subject a prophylactically effective amount of ananti-idiotype antibody to the polypeptide of claim 11 and apharmaceutically acceptable carrier.
 25. A method of treating aStreptococcus pneumoniae infection in a subject, comprisingadministering to the subject a therapeutically effective amount of theantibody of claim 12 and a pharmaceutically acceptable carrier.
 26. Amethod of treating a Streptococcus pneumoniae infection in a subject,comprising administering to the subject a therapeutically effectiveamount of the antibody of claim 13 and a pharmaceutically acceptablecarrier.